ginS

ABSTRACT

The invention provides ginS polypeptides and, polynucleotides encoding ginS polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ginS polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the ginS (autoinducersynthesis) family, as well as their variants, herein referred to as“ginS,” “ginS polynucleotide(s),” and “ginS polypeptide(s)” as the casemay be.

BACKGROUND OF THE INVENTION

[0002] It is particularly preferred to employ genes and gene productsfrom Porphyromonas gingivalis as targets for the development ofantibiotics. Porphyromonas gingivalis is a clinically importantmicroorgansim found within the human oral cavity gingival withincrevicular fluid, as a member of the subgingival plaque, on the tongue,tonsils, and pharynx (Mayrand, et al., Micro. Rev. 52: 134-152 (1988).Porphyromonas gingivalis is a non-motile, Gram negative anaerobicbacterium involved in the generation of breath malodour and stronglyimplicated in causing periodontal disease in humans. Recent evidencealso demonstrates a clear correlation between systemic exposure toPorphyromonas gingivalis with cardiovascular disease, diabetes and anincreased likelihood of pre-term births (up to a 7× risk increase forspontaneous pre-term births, possibly caused by by-products of bacterialmetabolism which induce the release of mediators linked to theinitiation of labour) and low neonatal birth weight (18% of low birthweights may be attributable to periodonitis) (Slots, et al. J. Clin.Periodontol. 13: 570-577 (1986); Maiden, et al., J. Clin. Periodontol.17: 1-13 (1990); Offenbacher, et al., J. Periodontol. 67: 1103-1113(1996)). Porphyromonas gingivalis elaborates a large number of virulencefactors including fimbriae (Lamont, et al., Oral Micro. Immunol. 8:272-276 (1993); Miyata, et al., Infect. Immun. 65: 3515-3519 (1997);Pike, et al.,. J. Bacteriol. 178: 2876-2882 (1996); Weinberg, et al.,Infect. Immun. 65: 313-316 (1997); Yoneda, et al. Oral Microbiol.Immunol. 11: 129-134 (1996)) and two major classes of proteases, KGP(Lys-X gingipain) and RGP (Arg-X gingipain) (Kuramitsu, et al., J.Periodontol. Res. 32: 140-142 (1997); Nakayama, et al., J. Biol. Chem.270: 23619-23626 (1995); Okamoto, et al., J. Biochem. 120: 398-406(1996); Park, et al., Infect. Immun. 61: 4139-4146 (1993)), known to beinvolved in mediating cellular attachment, tissue invasion, destruction(Birkedal-Hansen, et al., J Periodontal Res. 23: 258-64 (1988); Kato, etal., J. Bacteriol. 174: 3889-3895 (1992); Lantz, et al., J. Bacteriol.173: 495-504 (1991)) and immune evasion (Imamura, et al., J. Clin.Invest. 94; 361-367 (1994); Nilsson, et al., Infect. Immun. 50: 467-471(1985)). It has been shown in several other bacteria including Aeromonashydrophila (Swift, et al., TIBS 21: 214-219 (1996). Envinia carotovora(Bainton, et al., Gene, 116: 87-91 (1992); Jones, et al., EMBO J. 12:2477-2482 (1993)) and Pseudomonas aeruginosa (Gambello, et al., J.Bacteriol. 173: 3000-3009 (1991); Latifi, et al., Mol. Micro. 17:333-343 (1995); Pearson, et al., J. Bacteriol. 179: 5756-5767 (1997)),that extracellular protease activity is controlled by the celldensity-dependent system known as quorum sensing. Many bacteria use amechanism homologous to that of Vibrio fischeri in which the signallingmolecule (or autoinducer) is an N-acylhomoserine lactone (AHL)synthesized by luxI and responded to by direct binding to thetranscriptional regulator luxR (Williams, et al., FEMS Micro. Lett. 100:161-168 (1992); Salmond, et al., Mol. Microbiol. 16: 615-624 (1995);Swift, et al., Berlin Heidelberg: Springer-Verlag, pp.185-207 (1998);Stevens, et al., J. Bacteriol. 179: 557-562 (1997)). A diverse number ofphenotypes are regulated in this way, including bioluminescence,motility and expression of virulence determinants (Bainton, et al.,Biochem. J. 288: 997-1004 (1992); Bainton, et al., Gene, 116: 87-91(1992).

[0003] In the bioluminescent bacterium Vibrio harveyi, two independentquorum sensing systems are found which are controlled by a number ofproteins, none of which are homologous to the luxR/I proteins fromVibrio fischeri (Cao, et al., J. Bacteriol. 175: 3856-3862 (1993),Bassler, et al., Mol. Micro. 12: 403-412 (1994a), Bassler, et al., Mol.Micro. 13: 273-286 (1994b), Freeman, et al., Mol. Micro. 31: 665-677(1999a), Freeman, et al., J. Bacteriol. 181: 899-906 (1999). One system,however, does involve the synthesis of an AHL (3-OH-C4-HSL) by thesignal generators luxLM. Once a critical concentration is reached,3-OH-C4-HSL interacts with the response regulator luxN. The secondsystem requires luxS, homologues of which were identified in a number ofbacteria including Escherichia coli, Salmonella typhimurium, Bacillussubtilis and Helicobacter pylori (Surette, et al., Proc. Natl. Acad.Sci, USA. 96: 1639-1644 (1999a), Surette, et al., Mol. Micro. 31:585-595 (1999b), Joyce, et al., J. Bacteriol. 182: 3638-3643 (2000).

[0004] LuxS is involved in the production of a signalling molecule ofunknown chemical structure, AI-2, which interacts with responseregulators, luxPQ. Information from both quorum sensing systems isrelayed to the two-component regulator luxO, via a phospho-relayprotein, luxU. Dephosphorylation of luxO results in activation ofluminescence (Freeman, et al., Mol. Micro. 31: 665-677 (1999a), Freeman,et al., J. Bacteriol. 181: 899-906 (1999b).

[0005] It is thought that luxS is involved in bacterial cross-talk sinceit is present in both Gram-positive and Gram-negative bacteria (Surette,et al., Proc. Natl. Acad. Sci, USA. 96: 1639-1644 (1999a), Surette, etal.,. Mol. Micro. 31: 585-595 (1999b), and it has been reported toinfluence type III secretion in enterovirulent Escherichia coli(Sperandio, et al., PNAS 96: 15196-15201 (1999).

[0006] Breakdown of tissue proteins is an essential feature of thepathogenesis of periodontal disease and similarly, protein breakdown ofdietary proteins is a critical step leading to breath malodour. Bacteriasuch as Porphyromonas gingivalis and Prevotella intermedia must breakdown host gingival connective tissue in order to replicate and causedisease; breakdown products from protein include volatile sulphurcompounds, principally H₂S and methylmercaptan and also diamines such asputrescine and cadaverine, which are among the main causes of badbreath. Many of the important odourigenic anaerobes present in the oralcavity e.g, Fusobacterium nucleatum, Haemophilus segnis, Haemophilusparainfluenzae, Veillonella alcalescens and Veillonella parvula generateodorous compounds when supplied with a free amino acids e.g, cysteine,methionine, tryptophan, omithine and arginine, but are themselves unableto break down longer chain polypeptides. It has been proposed that theproteolytic activity of Porphyromonas gingivalis, particularlydipeptidyl peptidase and deaminase activity, is central to the abilityof this organism to break down dietary proteins, which then liberatefree amino-acids subsequently metabolised by other plaque bacteriaresulting in the development of breath malodour.

[0007] Because of the difficulty in mechanically removing the anaerobicplaque from interdental areas and from the tongue dorsum and also,because of the predisposition of chlorhexidine (the active ingredientfound in mouthrinses commonly employed to treat gingivitis and restoreoral hygiene) to be bitter-tasting and to cause extrinsic staining ofteeth, there is a clear unment clinical need for polynucleotides andpolypeptides, such as the ginS embodiments of the invention, that have apresent benefit of, among other things, being useful to screen compoundsfor antimicrobial activity and/or the ability to inhibit the proteolyticand/or deaminative activity associated with this organism which could bedeveloped as dentifrices, mouthrinses, dental lozenges and/or dentalgums.

[0008] Although many different bacterial genera have been found to beassociated with periodontal disease, Porphyromonas gingivalis is nowknown to be one of the critical bacteria species involved in theprogression of gingivitis and the invasive infection of the host tissuearising from the periodontal pocket, largely because of its' significantproteolytic activity.

[0009] Several forms of periodontal disease are now recognised, basedupon the presence or absence of inflammation, extent and pattern ofattachment loss, probing pocket depth, patient's age at onset, rate ofprogression, and presence of various signs and symptoms e.g, pain andulceration. However, all share key common features in that the diseaseaffects the periodontium or supporting structures of the teeth includinggingiva, periodontal ligament, and alveolar bone. Bacterial infection isthe primary etiological factor causing periodontal disease, and themajority of these diseases are inflammatory lesions caused by theaccumulation of microorganisms around the gingival margin.

[0010] Gingivitis defines inflammation that is confined to the gingiva,while periodontitis is characterized by subsequent destruction of boneand periodontal ligament resulting in loss of attachment to the tooth.Periodontitis is defined as a chronic inflammatory disease of theperiodontium occurring in response to bacterial plaque on the adjacentteeth; characterized by gingivitis, destruction of the alveolar bone andperiodontal ligament, apical migration of the epithelial attachmentresulting in the formation of periodontal pockets, and ultimatelyloosening and exfoliation of the teeth.

[0011] One hypothesis is that virulence gene expression in Porphyromonasgingivalis may be regulated via the production of molecules related tothe AHL/Lux quorum sensing system, employed by several species ofnon-oral Gram -ve bacteria. To date, no homologues of AHL-basedsignalling system have been identified in oral bacteria. However, ginswas first identified by homology with the luxS gene of Vibrio harveyiwhich encodes a soluble signalling molecule, AI-2.

[0012] Periodontal disease is currently by surgical excision of diseasedtissue either alone or in combination with systemic or locally actingantibitoics, particularly the tetracyclines. In severe cases, this mayneed to be followed by reconstructive surgery. However, given theconcern surrounding the use of antibitoics and the generation ofantibiotic-resistant clinical isolates, the issues surrounding the useof tetratcyclines in children (tetracyclines become incorporated intodeveloping teeth and bones) and the difficulties in delivering andmaintaining effective drug levels locally e.g, from polymeric chips orsurgically-implanted sutures, it is clear that there is an unmet medicalneed and demand for new anti-microbial agents, vaccines, drug screeningmethods, and diagnostic tests targeted at Porphyromonas gingivalis.

[0013] Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

[0014] Clearly, there exists a need for polynucleotides andpolypeptides, such as the ginS embodiments of the invention, that have apresent benefit of, among other things, being useful to screen compoundsfor antimicrobial activity. Such factors are also useful to determinetheir role in pathogenesis of infection, dysfunction and disease. Thereis also a need for identification and characterization of such factorsand their antagonists and agonists to find ways to prevent, ameliorateor correct such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

[0015] The present invention relates to ginS, in particular ginSpolypeptides and ginS polynucleotides, recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, includingtreatment of microbial diseases, amongst others. In a further aspect,the invention relates to methods for identifying agonists andantagonists using the materials provided by the invention, and fortreating microbial infections and conditions associated with suchinfections with the identified agonist or antagonist compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with microbial infections and conditionsassociated with such infections, such as assays for detecting ginSexpression or activity.

[0016] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 shows V.harveyi bioluminescence assay demonstratingcomplementation of E.coli DH5α luxS mutation by pMALGin1. FIG. 1 furthershows the V.harveyi luminescence assay uses overnight culturesupernatants demonstrating complementation of the E.coli DH5α luxS_(EC)mutation in DH5α expressing gins (GinS1 & GinS2) and reducedbioluminescence of two ginS⁻ null mutants (Mut1 & Mut2). Negativecontrol=E.coli DH5α; Positive control=V.harveyi BB120.

[0018]FIG. 2 shows the same data exhibited in FIG. 1 expressed as ahistogram.

[0019]FIG. 3 shows SDS-PAGE analysis demonstrating expression ofMalE-GinS protein fusion. Further, FIG. 3 shows SDS-PAGE analysisshowing the expressed MalE-GinS protein fusion. pMAL-c2 was induced with0.3 mM IPTG. Lane 1 shows DH5-α; lane 2 MalE; lane 3 MalE-GinS. Lanes1-3 are soluble; lanes 4-6 are the same as 1-3 but are insolublefractions.

[0020]FIG. 4 shows MalE-GinS fusion construct induces expression ofbiologically active AI-2 in E.coli DH5α, dompelementing the mutation inthis strain

[0021]FIG. 5 shows purification of the MalE-GinS fusion protein byaffinity chromatography.

[0022]FIG. 6 shows SDS-PAGE analysis of fractions collected fromaffinity chromotography column.

[0023]FIG. 7 shows SDS-PAGE analysis showing Factor Xa cleavage of GinSfrom the MalE fusion protein. Further, FIG. 7 shows SDS-PAGE analysisshowing cleavage of GinS from MalE following incubation with Factor Xaat RT. Lanes 1 & 2 show uncut fusion; lane 3: 2 hours incubation; lane4: 4 hours incubation; lane 5: 6 hours incubation; lane 6: 22 hoursincubation.

[0024]FIG. 8 shows production of AI-2 and MalE-GinS throughout thegrowth cycle in E.coli This data demonstrates that AI-2 production peaksbetween 7 and 9 hours of the growth curve. This coincides with ginsexpression (see Western blot inset), which appears to be maximal by 5hours and is maintained through to 24 hours of the growth curve. Thistemporal link would fit with the model that ginS is required forexpression of AI-2.

[0025]FIG. 9 shows production of AI-2 and expression of MalE-GinSthroughout growth of P. gingivalis. Western blots were carried out usinga polyclonal antibody to probe GinS from cell lysates of the wild-typeP.gingivalis W50 (Panel A) and the ginS⁻ null mutant (Panel B)throughout the growth curve. Lane 1 show Positive GinS control; Lanes2-7 show cell lysates at 6, 12, 24, 72 & 96 hours of growth.

[0026]FIG. 10 shows confirmation of creation of insertional ‘Null’mutation in ginS in P.gingivalis W50 by Agarose Gel ElectrophoreticAnalysis of PCR Amplification of erm Cassette.

[0027]FIG. 11 shows Southern blot analysis demonstrating chromosomalintegration of the erm Cassette in the P.gingivalis ginS⁻ Null Mutant.The control (lane 1) demonstrates hybridization of probe to the ginSgene amplified from a plasmid. Lanes 2 & 3 demonstrate hybridization toginS sequences amplified from the P.gingivalis chromosome. Prior toSouthern blotting, DNA was cut with a restriction enzyme with twocleavage sites within ginS. This results in two hybridizing bands in thewild-type but only a single in the ginS⁻ null mutant, due to eliminationof the internal restriction site following replacement with the ermcassette.

[0028]FIG. 12 shows comparison of Total Protease Expression in Wild-typeand ginS⁻ Null Mutant Culture Supernatants by Zymography

[0029]FIG. 13 shows Expression of Kgp (Lys-X gingipain) and RgpA (Arg-Xgingipain) Proteases in Wildtype P.gingivalis Strain W50 Compared to theginS⁻ Mutant by SDS-PAGE Analysis of Total Protein Lysates, Probed withSpecific Antibodies to Kgp & RgpA.

[0030]FIG. 14 shows determination of KGP and RGP Protease Activity inWhole Cell Lysates and Culture Supernatants of P.gingivalis W50 and theginS⁻ Null Mutant.

[0031]FIG. 15 shows Total Rgp (Arg-X Gingipain) Activity in Soluble CellFractions of Wildtype P.gingivalis and the ginS⁻ Null Mutant Over Time.

[0032]FIG. 16 shows total Rgp (Arg-X Gingipain) Activity in CultureSupernatants from Wildtype P.gingivalis and the ginS⁻ Null Mutant OverTime.

[0033]FIG. 17 shows determination of Haemagglutinin Activity in ginS⁻Null Mutant. In accordance with the direct structural relationship ofproteases and haemagglutinins in P.gingivalis (Yoneda et al, 1996), a4-fold decrease in agglutination of sheep erythrocytes was observed withthe ginS⁻ mutant of P.gingivalis, further confirming a decrease inproduction of proteases.

[0034]FIG. 18 shows 2-D Gel Electrophoresis of Total Protein Lysatesfrom Wild-type P.gingivalis W50 and ginS⁻ Null Mutant

DESCRIPTION OF THE INVENTION

[0035] The invention relates to ginS polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of ginS from Porphyromonasgingivalis, that is related by amino-acid sequence homology to the luxSpolypeptide of Borrelia Bergdorferi (ATCC 35210). The invention relatesespecially to ginS having a nucleotide and amino-acid sequences set outin Table 1 as SEQ ID NO:1 and SEQ ID NO:2 respectively. Note thatsequences recited in the Sequence Listing below as “DNA” represent anexemplification of the invention, since those of ordinary skill willrecognize that such sequences can be usefully employed inpolynucleotides in general, including ribopolynucleotides. TABLE 1 GinSPolynucleotide and ginS Polypeptide Sequences (A) Porphyromonasgingivalis W50 ginS polynucleo- tide coding sequence. [SEQ ID NO:1]5′-ATGGAAAAAATTCCCAGTTTTCAGTTAGATCATATTCGCCTCAAACGAGGCATATATGTCTCCCGCAAGGACTATATAGGGGGAGAGGTGGTTACGACTTTCGATATTCGAATGAAAGAGCCCAATCGCGAACCGGTGCTTGGGGCACCCGAACTGCATACGATCGAGCATTTGGCTGCAACTTATCTGCGTAATCATCCGCTTTATAAGGACAGGATCGTTTTCTGGGGGCCGATGGGCTGCCTTACGGGCAATTACTTTCTGATGCGAGGCGATTACGTATCCAAAGATATACTGCCCCTCATGCAGGAGACTTTCCGCTTCATCAGAGACTTCGAAGGAGAAGTGCCGGGTACGGAGCCGCGCGACTGTGGCAACTGCCTGCTGCACAACCTGCCGATGGCCAAATATGAGGCCGAGAAATACCTGCGTGAGGTACTCGATGTAGCGACGGAGGAGAACCTGAACTATCCCGACTGA-3′ (B) Porphyromonas gingivalis W50ginS polypeptide sequence deduced from a polynucleotide sequence in thistable. [SEQ ID NO:2] NH₂-MEKIPSFQLDHIRLKRGIYVSRKDYIGGEVVTTFDIRMKEPNREPVLGAPELHTIEHLAATYLRNHPLYKDRIVFWGPMGCLTGNYFLMRGDYVSKDILPLMQETFRFIRDFEGEVPGTEPRDCGNCLLHNLPMAKYEAEKYLREV LDVATEENLNYPD.-COOH

[0036]Porphyromonas gingivalis strain W50 (Shah, et al., Oral MicrobiolImmunol 4:19-23 (1989) comprises a full length ginS gene. The sequenceof the polynucleotides comprised in this strain, as well as the aminoacid sequence of any polypeptide encoded thereby, are controlling in theevent of any conflict with any description of sequences herein.

[0037] In one aspect of the invention there is provided an isolatednucleic acid molecule encoding a mature polypeptide expressible by thePorphyromonas gingivalis W50 strain, which polypeptide is comprised inthis original strain strain. Further provided by the invention are ginSpolynucleotide sequences in the original strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare ginS polypeptide and polynucleotide sequences isolated from theoriginal strain.

[0038] Polypeptides

[0039] ginS polypeptide of the invention is substantiallyphylogenetically related to other proteins of the luxS (autoinducersynthesis) family.

[0040] In one aspect of the invention there are provided polypeptides ofPorphyromonas gingivalis referred to herein as “ginS” and “ginSpolypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0041] Among the particularly preferred embodiments of the invention arevariants of ginS polypeptide encoded by naturally occurring alleles of aginS gene.

[0042] The present invention further provides for an isolatedpolypeptide that: (a) comprises or consists of an amino acid sequencethat has at least 95% identity, most preferably at least 97-99% or exactidentity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2;(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence that has at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO:1over the entire length of SEQ ID NO:1; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

[0043] The polypeptides of the invention include a polypeptide of Table1 [SEQ ID NO:2] (in particular a mature polypeptide) as well aspolypeptides and fragments, particularly those that has a biologicalactivity of ginS, and also those that have at least 95% identity to apolypeptide of Table 1 [SEQ ID NO:2] and also include portions of suchpolypeptides with such portion of the polypeptide generally comprisingat least 30 amino-acids and more preferably at least 50 amino-acids.

[0044] The invention also includes a polypeptide consisting of orcomprising a polypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0045] wherein, at the amino terminus, X is hydrogen, a metal or anyother moiety described herein for modified polypeptides, and at thecarboxyl terminus, Y is hydrogen, a metal or any other moiety describedherein for modified polypeptides, R₁ and R₃ are any amino acid residueor modified amino acid residue, m is an integer between 1 and 1000 orzero, n is an integer between 1 and 1000 or zero, and R₂ is an aminoacid sequence of the invention, particularly an amino acid sequenceselected from Table 1 or modified forms thereof. In the formula above,R₂ is oriented so that its amino terminal amino acid residue is at theleft, covalently bound to R₁, and its carboxy terminal amino acidresidue is at the right, covalently bound to R₃. Any stretch of aminoacid residues denoted by either R₁ or R₃, where m and/or n is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer. Other preferred embodiments of the invention are providedwhere m is an integer between 1 and 50, 100 or 500, and n is an integerbetween I and 50, 100, or 500.

[0046] It is most preferred that a polypeptide of the invention isderived from Porphyromonas gingivalis, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0047] A fragment is a variant polypeptide having an amino acid sequencethat is entirely the same as part but not all of any amino acid sequenceof any polypeptide of the invention. As with ginS polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region in a single larger polypeptide.

[0048] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], orof variants thereof, such as a continuous series of residues thatincludes an amino- and/or carboxyl-terminal amino acid sequence.Degradation forms of the polypeptides of the invention produced by or ina host cell, particularly a Porphyromonas gingivalis, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

[0049] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2,or an isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

[0050] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

[0051] Polynucleotides

[0052] It is an object of the invention to provide polynucleotides thatencode ginS polypeptides, particularly polynucleotides that encode apolypeptide herein designated ginS.

[0053] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding ginS polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] that includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Porphyromonas gingivalis.

[0054] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing ginS polypeptides andpolynucleotides, particularly Porphyromonas gingivalis ginS polypeptidesand polynucleotides, including, for example, unprocessed RNAs, nrbozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

[0055] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes aginS polypeptide having a deduced amino-acid sequence of Table 1 [SEQ IDNO:2] and polynucleotides closely related thereto and variants thereof.

[0056] In another particularly preferred embodiment of the inventionthere is a ginS polypeptide from Porphyromonas gingivalis comprising orconsisting of an amino-acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

[0057] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NO:1], a polynucleotide of theinvention encoding ginS polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Porphyromonas gingivalisW50 cells as starting material, followed by obtaining a full lengthclone. For example, to obtain a polynucleotide sequence of theinvention, such as a polynucleotide sequence given in Table 1 [SEQ IDNO:1], typically a library of clones of chromosomal DNA of Porphyromonasgingivalis W50 in Escherichia coli or some other suitable host is probedwith a radiolabeled oligonucleotide, preferably a 17-mer or longer,derived from a partial sequence. Clones carrying DNA identical to thatof the probe can then be distinguished using stringent hybridizationconditions. By sequencing the individual clones thus identified byhybridization with sequencing primers designed from the originalpolypeptide or polynucleotide sequence it is then possible to extend thepolynucleotide sequence in both directions to determine a full lengthgene sequence. Conveniently, such sequencing is performed, for example,using denatured double stranded DNA prepared from a plasmid clone.Suitable techniques are described by Maniatis, T., Fritsch, E. F. andSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). (see inparticular Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Direct genomic DNA sequencing mayalso be performed to obtain a full length gene sequence. Illustrative ofthe invention, each polynucleotide set out in Table 1 [SEQ ID NO: 1] wasdiscovered in a DNA library derived from Porphyromonas gingivalis.

[0058] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1]contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO:2] with adeduced molecular weight that can be calculated using amino-acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 469 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

[0059] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of: (a) apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1; (b) a polynucleotide sequence encoding apolypeptide that has at least 95% identity, even more preferably atleast 97-99% or 100% exact, to the amino acid sequence of SEQ ID NO:2,over the entire length of SEQ ID NO:2.

[0060] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Porphyromonasgingivalis, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

[0061] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in Table 1[SEQ ID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0062] A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 469 set forth in SEQ ID NO:1 ofTable 1, both of that encode a ginS polypeptide.

[0063] The invention also includes a polynucleotide consisting of orcomprising a polynucleotide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0064] wherein, at the 5′ end of the molecule, X is hydrogen, a metal ora modified nucleotide residue, or together with Y defines a covalentbond, and at the 3′ end of the molecule, Y is hydrogen, a metal, or amodified nucleotide residue, or together with X defines the covalentbond, each occurrence of R₁ and R₃ is independently any nucleic acidresidue or modified nucleic acid residue, m is an integer between 1 and3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is anucleic acid sequence or modified nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1 ora modified nucleic acid sequence thereof. In the polynucleotide formulaabove, R₂ is oriented so that its 5′ end nucleic acid residue is at theleft, bound to R₁, and its 3′ end nucleic acid residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either R₁and/or R₂, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Where, in apreferred embodiment, X and Y together define a covalent bond, thepolynucleotide of the above formula is a closed, circularpolynucleotide, that can be a double-stranded polynucleotide wherein theformula shows a first strand to which the second strand iscomplementary. In another preferred embodiment m and/or n is an integerbetween 1 and 1000. Other preferred embodiments of the invention areprovided where m is an integer between 1 and 50, 100 or 500, and n is aninteger between 1 and 50, 100, or 500.

[0065] It is most preferred that a polynucleotide of the invention isderived from Porphyromonas gingivalis, however, it may preferably beobtained from other organisms of the same taxonomic genus. Apolynucleotide of the invention may also be obtained, for example, fromorganisms of the same taxonomic family or order.

[0066] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Porphyromonas gingivalis ginShaving an amino-acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

[0067] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

[0068] Further particularly preferred embodiments are polynucleotidesencoding ginS variants, that have the amino acid sequence of ginSpolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified,deleted and/or addes, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of ginS polypeptide.

[0069] Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

[0070] Further preferred embodiments of the invention arepolynucleotides that are at least 95% or 97% identical over their entirelength to a polynucleotide encoding ginS polypeptide having an aminoacid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides thatare complementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

[0071] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

[0072] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to ginS polynucleotidesequences, such as those polynucleotides in Table 1.

[0073] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 533 Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0074] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library comprising a complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO:1 or a fragment thereof; and isolatingsaid polynucleotide sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers fully describedelsewhere herein.

[0075] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding ginS and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to a ginS gene. Suchprobes generally will comprise at least 15 nucleotide residues or basepairs. Preferably, such probes will have at least 30 nucleotide residuesor base pairs and may have at least 50 nucleotide residues or basepairs. Particularly preferred probes will have at least 20 nucleotideresidues or base pairs and will have lee than 30 nucleotide residues orbase pairs.

[0076] A coding region of a ginS gene may be isolated by screening usinga DNA sequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

[0077] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., Proc. Natl. Acad. Sci. USA85: 8998-9002 (1988)). Recent modifications of the technique,exemplified by the Marathon™ technology (Clontech Laboratories Inc.) forexample, have significantly simplified the search for longer cDNAs. Inthe Marathon™ technology, cDNAs have been prepared from mRNA extractedfrom a chosen tissue and an ‘adaptor’ sequence ligated onto each end.Nucleic acid amplification (PCR) is then carried out to amplify the“missing” 5′ end of the DNA using a combination of gene specific andadaptor specific oligonucleotide primers. The PCR reaction is thenrepeated using “nested” primers, that is, primers designed to annealwithin the amplified product (typically an adaptor specific primer thatanneals further 3′ in the adaptor sequence and a gene specific primerthat anneals further 5′ in the selected gene sequence). The products ofthis reaction can then be analyzed by DNA sequencing and a full-lengthDNA constructed either by joining the product directly to the existingDNA to give a complete sequence, or carrying out a separate full-lengthPCR using the new sequence information for the design of the 5′ primer.

[0078] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0079] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0080] The invention also provides polynucleotides that encode apolypeptide that is a mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to a maturepolypeptide (when a mature form has more than one polypeptide chain, forinstance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away froma mature protein by cellular enzymes.

[0081] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0082] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0083] As will be recognized, the entire polypeptide encoded by an openreading frame is often not required for activity. Accordingly, it hasbecome routine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

[0084] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

[0085] Vectors, Host Cells, Expression Systems

[0086] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0087] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

[0088] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal., Basic Methods In Molecular Biology (1986) and Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0089] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococciEscherichia coli, streptomyces, cyanobacteria, Bacillus subtilis, andStaphylococcus aureus; fungal cells, such as cells of a yeast,Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans andAspergillus; insect cells such as cells of Drosophila S2 and SpodopteraSf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 andBowes melanoma cells; and plant cells, such as cells of a gymnosperm orangiosperm.

[0090] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., Molecular Cloning: ALaboratory Manual (1989).

[0091] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0092] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0093] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0094] This invention is also related to the use of ginS polynucleotidesand polypeptides of the invention for use as diagnostic reagents.Detection of ginS polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a human, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the ginS gene or ginS protein, maybe detected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

[0095] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled ginS polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, VI and SI protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

[0096] In another embodiment, an array of oligonucleotides probescomprising ginS nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science 274: 610 (1996).

[0097] Thus in another aspect, the present invention relates to adiagnostic kit that comprises: (a) a polynucleotide of the presentinvention, preferably the nucleotide sequence of SEQ ID NO:1, or afragment thereof; (b) a nucleotide sequence complementary to that of(a); (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody toa polypeptide of the present invention, preferably to the polypeptide ofSEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c)or (d) may comprise a substantial component. Such a kit will be of usein diagnosing a disease or susceptibility to a Disease, among others.

[0098] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0099] The differences in a polynucleotide and/or polypeptide sequencebetween organisms possessing a first phenotype and organisms possessinga different, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

[0100] Cells from an organism carrying mutations or polymorphisms(allelic variations) in a polynucleotide and/or polypeptide of theinvention may also be detected at the polynucleotide or polypeptidelevel by a variety of techniques, to allow for serotyping, for example.For example, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to use RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR. As an example, PCRprimers complementary to a polynucleotide encoding ginS polypeptide canbe used to identify and analyze mutations. The invention furtherprovides these primers with 1, 2, 3 or 4 nucleotides removed from the 5′and/or the 3′ end. These primers may be used for, among other things,amplifying ginS DNA and/or RNA isolated from a sample derived from anindividual, such as a bodily material. The primers may be used toamplify a polynucleotide isolated from an infected individual, such thatthe polynucleotide may then be subject to various techniques forelucidation of the polynucleotide sequence. In this way, mutations inthe polynucleotide sequence may be detected and used to diagnose and/orprognose the infection or its stage or course, or to serotype and/orclassify the infectious agent.

[0101] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byPorphyromonas gingivalis, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a ginS polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

[0102] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of ginS polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a ginS polypeptide, in a sample derived from a host, such as abodily material, are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis, antibody sandwich assays, antibody detection and ELISAassays.

[0103] Antagonists and Agonists—Assays and Molecules

[0104] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

[0105] Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g.,inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc, as the case may be, ofginS polypeptides and polynucleotides; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2): Chapter 5 (1991)).

[0106] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists, in the absence of an agonist orantagonist, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution comprising apolypeptide or polynucleotide of the present invention, to form amixture, measuring ginS polypeptide and/or polynucleotide activity inthe mixture, and comparing the ginS polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and ginS polypeptide, as herein described, can alsobe used for high-throughput screening assays to identify antagonists ofthe polypeptide of the present invention, as well as of phylogeneticallyand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270:9459-9471 (1995)).

[0107] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents that may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0108] The invention also provides a method of screening compounds toidentify those that enhance (agonist) or block (antagonist) the actionof ginS polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising ginS polypeptide and a labeled substrate or ligandof such polypeptide is incubated in the absence or the presence of acandidate molecule that may be a ginS agonist or antagonist. The abilityof the candidate molecule to agonize or antagonize the ginS polypeptideis reflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of ginS polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in ginS polynucleotide or polypeptideactivity, and binding assays known in the art.

[0109] Polypeptides of the invention may be used to identify membranebound or soluble receptors, if any, for such polypeptide, throughstandard receptor binding techniques known in the art. These techniquesinclude, but are not limited to, ligand binding and crosslinking assaysin which the polypeptide is labeled with a radioactive isotope (forinstance, ¹²⁵I), chemically modified (for instance, biotinylated), orfused to a peptide sequence suitable for detection or purification, andincubated with a source of the putative receptor (e.g., cells, cellmembranes, cell supernatants, tissue extracts, bodily materials). Othermethods include biophysical techniques such as surface plasmon resonanceand spectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

[0110] The fluorescence polarization value for a fluorescently-taggedmolecule depends on the rotational correlation time or tumbling rate.Protein complexes, such as formed by ginS polypeptide associating withanother ginS polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

[0111] Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of ginS polypeptide dimers,trimers, tetramers or higher order structures, or structures formed byginS polypeptide bound to another polypeptide. ginS polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

[0112] Surface plasmon resonance can be used to monitor the effect ofsmall molecules on ginS polypeptide self-association as well as anassociation of ginS polypeptide and another polypeptide or smallmolecule. ginS polypeptide can be coupled to a sensor chip at low sitedensity such that covalently bound molecules will be monomeric. Solutionprotein can then passed over the ginS polypeptide -coated surface andspecific binding can be detected in real-time by monitoring the changein resonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for ginS polypeptideself-association as well as an association of ginS polypeptide andanother polypeptide or small molecule.

[0113] A scintillation proximity assay may be used to characterize theinteraction between an association of ginS polypeptide with another ginSpolypeptide or a different polypeptide ginS polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled ginSpolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon ginS polypeptide binding and compounds that prevent ginSpolypeptide self-association or an association of ginS polypeptide andanother polypeptide or small molecule will diminish signal.

[0114] In other embodiments of the invention there are provided methodsfor identifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

[0115] Another example of an assay for ginS agonists is a competitiveassay that combines ginS and a potential agonist with ginS-bindingmolecules, recombinant ginS binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. ginS can be labeled, such as byradioactivity or a calorimetric compound, such that the number of ginSmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0116] It will be readily appreciated by the skilled artisan that apolypeptide and/or polynucleotide of the present invention may also beused in a method for the structure-based design of an agonist orantagonist of the polypeptide and/or polynucleotide, by: (a) determiningin the first instance the three-dimensional structure of the polypeptideand/or polynucleotide, or complexes thereof; (b) deducing thethree-dimensional structure for the likely reactive site(s), bindingsite(s) or motif(s) of an agonist or antagonist; (c) synthesizingcandidate compounds that are predicted to bind to or react with thededuced binding site(s), reactive site(s), and/or motif(s); and (d)testing whether the candidate compounds are indeed agonists orantagonists. It will be further appreciated that this will normally bean iterative process, and this iterative process may be performed usingautomated and computer-controlled steps.

[0117] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, a Disease, relatedto either an excess of, an under-expression of, an elevated activity of,or a decreased activity of ginS polypeptide and/or polynucleotide.

[0118] If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the ginSpolypeptide and/or polypeptide.

[0119] In still another approach, expression of the gene encodingendogenous ginS polypeptide can be inhibited using expression blockingtechniques. This blocking may be targeted against any step in geneexpression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O'Connor, J Neurochem 56: 560 (1991) inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res 6:3073 (1979); Cooney et al., Science 241:456(1988); Dervan et al., Science 251:1360 (1991)). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0120] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgamo orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0121] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial ginS proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

[0122] In accordance with yet another aspect of the invention, there areprovided ginS agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

[0123] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0124]Helicobacter pylori (herein “H.pylori”) bacteria infect thestomachs of over one-third of the world's population causing stomachcancer, ulcers, and gastritis (International Agency for Research onCancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori(International Agency for Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H.pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofginS polypeptides and/or polynucleotides) found using screens providedby the invention, or known in the art, particularly narrow-spectrumantibiotics, should be useful in the treatment of H.pylori infection.Such treatment should decrease the advent of H.pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

[0125] Glossary

[0126] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0127] “Bodily material(s) means any material derived from an individualor from an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

[0128] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, disease, such as, infections ofthe upper respiratory tract (e.g, otitis media, bacterial tracheitis,acute epiglottitis, thyroiditis), lower respiratory (e.g, empyema, lungabscess), cardiac (e.g, infective endocarditis), gastrointestinal (e.g.,secretory diarrhoea, splenic absces, retroperitoneal abscess), CNS(e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g, epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g, septic arthritis,osteomyelitis).

[0129] “Host cell(s)” is a cell that has been introduced (e.g,transformed or transfected) or is capable of introduction (e.g,transformation or transfection) by an exogenous polynucleotide sequence.

[0130] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990)). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)). The well knownSmith Waterman algorithm may also be used to determine identity.

[0131] Parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J Mol Biol. 48: 443-453(1970)). Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff,Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)).

[0132] Gap Penalty: 12

[0133] Gap Length Penalty: 4

[0134] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0135] Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970).

[0136] Comparison matrix: matches=+10, mismatch=0

[0137] Gap Penalty: 50

[0138] Gap Length Penalty: 3

[0139] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0140] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0141] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0142] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

[0143] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 95, 97 or 100%identity to a polypeptide reference sequence of SEQ ID NO:2, whereinsaid polypeptide sequence may be identical to the reference sequence ofSEQ ID NO:2 or may include up to a certain integer number of amino acidalterations as compared to the reference sequence, wherein saidalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of amino acid alterations is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)—(x _(a) ·y),

[0144] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

[0145] “Individual(s)” means a multicellular eukaryote, including, butnot limited to a metazoan, a mammal, an ovid, a bovid, a simian, aprimate, and a human.

[0146] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0147] “Organism(s)” means a (i) prokaryote, including but not limitedto, a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterni, Shigella flexneri,Pseudomonas aerutginosa, Franscisella tularensis, Bricella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii, Porphyromonas gingivalis and Chlamydiatrachomitis, (ii) an archaeon, including but not limited toArchaebacter, and (iii) a unicellular or filamentous eukaryote,including but not limited to, a protozoan, a fungus, a member of thegenus Saccharomyces, Kluveromyces, or Candida, and a member of thespecies Saccharomyces ceriviseae, Kluveromyces lactis, or Candidaalbicans.

[0148] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0149] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may compriseamino acids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may comprise many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, Proteins: Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in Post-translational Covalent Modification of Proteins, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

[0150] “Recombinant expression system(s)” refers to expression systemsor portions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

[0151] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled altisans.

EXAMPLES

[0152] The examples below are carried out using standard techniques,that are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Bacterial Strain Selection, Plasmid Curation, LibraryProduction and DNA Sequencing

[0153]Porphyromonas gingivalis W50 was maintained on FastidiousAnaerobic Agar (LabM) plates containing 5% (v/v) horse blood (TCS,Buckingham, U.K.) at 37° C. in an anaerobic cabinet (MK3 Anaerobic WorkStation, Dow Scientific) equilibrated in 80% nitrogen/10% hydrogen/10%carbon dioxide. Liquid cultures of Porphyromonas gingivalis W50 weregrown anaerobically at 37° C. in Brain Heart Infusion (BHI) broth(Oxoid) containing 5 μg/ml (w/v) haemin. The wildtype and mutant strainsof Porphyromonas gingivalis W50 were maintained as frozen bead stocks(PRO-LAB Diagnostics) and stored at −80° C. Freshly isolated colonieswere used for each experiment and incubation on plates in the anaerobiccabinet was allowed to proceed for 4-5 days before inoculating intobroth. The Porphyromonas gingivalis W50 genomic library was constructedin pBluescript SK⁺ (Stratagene).

[0154] Other bacterial strains used in this study are listed in Table 2.

TABLE 2. Bacterial Strains and Plasmid Reporter Constructs used forAutoinducer Detection

[0155] TABLE 2 Bacterial Strains and Plasmid Reporter Constructs usedfor Autoinducer Detection. Plasmid/ Strain Description Source/Ref. P.gingivalis Wildtype strain Shah et al, 1989 W50 V. harveyi Wildtypestrain Bassler et al, BB120 1997 V. harveyi Mutant strain which respondsto AI-2 Greenberg et al, BB170 1979 V. harveyi Mutant strain whichresponds to AI-1 Greenberg et al, BB886 1979 CV026 Double Tn5 mutantderived from Winson et al, C. violaeum ATCC 31532; N-acyl-HSL 1994sensor, Km^(R), Hg^(R) pSB401 N-acyl HSL plasmid containing a Winson etal, luxRI′::CDA fusion in pACYC 184, 1998a, b Tet^(R) pSB536 N-acyl HSLplasmid containing an Swift et al, 1997 ahyRI′::luxCDABE fusion inpRK415 Amp^(R) pSB1142 N-acyl HSL plasmid containing a lasRI′::luxCDABEfusion in pRK415, Tet^(R) pSB1075 N-acyl HSL plasmid containing a Winsonet al, lasRI′::luxCDABE fusion in pUC18, 1998a, b Tet^(R)

[0156] All of the plasmid reporters introduced into E.coli JM109 weregrown at 37° C. in Luria-Bertani medium (LB), containing 10 gbacto-tryptone (Difco), 5 g yeast extract (Difco) and 5 g NaCl (Sigma)per litre. The V.harveyi strains were grown at 30° C. in AB medium, therecipe for which has been previously reported (Greenberg et al, 1979).Antibiotics were used at the following concentrations (mg/litre);ampicillin (Amp), 50; clindamycin (Cn), 5; erythromycin (Erm), 300;tetracycline (Tet), 10. DNA isolation, restriction digests andtransformation of E.coli was performed as described by Sambrook et al(1989). Bacterial chromosomal DNA was purified using the CTAB method.Probes for Southern blot analysis were labelled by using theDIG-labelling system of Boerhinger Mannheim.

[0157] The polynucleotide having a DNA sequence given in Table 1 [SEQ IDNO: 1] was obtained from a library of clones of chromosomal DNA ofPorphyromonas gingivalis in E.coli. The sequencing data from two or moreclones comprising overlapping Porphyromnonas gingivalis DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

[0158] Methods 1 and 2 below

[0159] Total cellular DNA is isolated from Porphyromonas gingivalis W50according to standard procedures and size-fractionated by either of twomethods.

[0160] Method 1

[0161] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

[0162] Method 2

[0163] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g, RsaI, PaII, AluI,Bsh1235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and Escherichiacoli infected with the packaged library. The library is amplified bystandard procedures.

Example 2 N-acyl Homoserine Lactone Assays

[0164] Chromobacterium (CV026) forward and reverse assays and thin-layerchromatography of concentrated culture supernatants using CV026 andbioluminescent reporters have been previously reported (McClean et al,1997; Swift et al, 1997; Winson et al, 1998). Screening for detection ofluminescence was carried out using a Luminograph LB980 photon imagingcamera (EG&G Berthold) according to the Manufacturer's instructions.

Example 3 Molecular Cloning of ginS Polynucleotide

[0165] PCR amplification of ginS from P.gingivalis W50 chromosomal DNAwas carried out using primers Por115′-GTATTATCAGCGGAATTCCCGGCGAAGGTCG-3′ [SEQ ID NO:3] and Por125′-GATACCGCCTCCGGATCCAATAATCCATCCGG-3′ [SEQ ID NO:4] which were designedto the P.gingivalis W83 genome sequence and contained created EcoRI andBamHI restriction sites respectively. The PCR product was purified usinga Qiagen PCR purification kit according to the manufacturer'sinstructions, ligated into pHG327, previously digested with EcoRI andBamHII, (Stewart et al, 1986) and electroporated into E.coli DH5α(Sambrook et al, 1989), thus creating pHGin1.

Example 4 Mutagenesis of ginS in E.coli DH5α

[0166] A deletion mutant of ginS was prepared by chimaeric PCR to remove77 amino-acids. Two PCR products were generated from chromosomal DNA,containing NotI restriction sites, one with primers Por11 and Por13B5′-GCGGCCGCCACCAAATGCTCGATCGTATGCCAG-3′ [SEQ ID NO:5] the other withprimers Por14B 5′-TGGCGGCCGCGCGTGAGGTACTCGATGTAGG-3′ [SEQ ID NO:6] andPor12. Subsequently, 1 μl of each of these purified PCR products servedas a template in a second PCR amplification using primers Por11 andPor12. The PCR product was digested with BamHI and EcoRI, purified,ligated into pHG327 (similarly digested) and electroporated into E.coliDH5α, to create pHGin2.

Example 5 Autoinducer-2 Assay

[0167] The AI-2 bioassay using the V.harveyi biosensor BB170 (sensor 1⁻,sensor 2⁺) has been reported previously (Greenberg et al, Arch. Micro.120: 87-91 (1979).

[0168]P.gingivalis W50 was grown in a chemically defined medium (Milneret al, FEMS Microbiol. Letts. 140: 125-130 (1996), E.coli DH5α in LBmedium and the V.harveyi strains in AB medium and cell-free culturesupernatants were added to V.harveyi BB170 suspension in AB medium at10% (v/v). AI-2 activity is reported as Relative Light Units (RLU),measured in an EG&G Wallac Victor luminometer. Following incubation forthe time indicated.

Example 6 Expression of ginS

[0169] PCR amplification of ginS from P.gingivalis W50 chromosomal DNAwas also carried out using primers Por19a5′-AGACAATCCCGAATTCGAGATGGAA-3′ [SEQ ID NO:7] and Por20a5′-TGAGAAATAGAGCGGATCCTAAGC-3′ [SEQ ID NO:8] which contained EcoRI andBamHI restriction sites respectively. The product was purified, ligatedinto pMal-c2 (New England BioLabs) using the EcoRI/BamHI sites andelectroporated into E.coli DH50α, thus creating pMalGin1. DH5α(pMalGin1) was grown to an A600 of 0.2, 0.3 mM IPTG was added and growthwas continued until an A600 of 1.2. The cells were harvested bycentrifugation at 10,000×g for 10 min, resuspended in PBS and sonicated.Insoluble material was removed by centrifugation at 4,000×g for 5 min.Purification was carried out by affinity chromatography using an amyloseresin column (New England BioLabs) and elution in 10 mM maltoseaccording to the manufacturer's instruction.

Example 7 TCA Precipitation and SDS-PAGE Analysis

[0170] Cultures of P.gingivalis wildtype (strain W50) and the ginSmutant strain were TCA precipitated by adding 10% (v/v) of 100% TCA(w/v) in H₂O and placing on ice for 1 h. Precipitated proteins wereharvested by centrifugation at 10,000×g for 15 min and washed with icecold acetone. Pellets were dried in air and resuspended in PBS. Sodiumdodecyl sulphate (SDS) sample buffer was added and the proteins wereseparated by SDS-Polyacrylamide Gel Electrophoresis (PAGE) on 12.5%gels.

Example 8 Purification of GinS for Antibody Production

[0171] Factor X_(a) enzyme (New England BioLabs) was diluted to 200μg/ml in PBS and 800 μg of purified MalE-ginS at a concentration of 1.2mg/ml was incubated with the enzyme at room temperature for either 2, 4,6 or 22 h, after which time, SDS sample buffer was added and the sampleswere analysed by SDS-PAGE. Purified and cleaved ginS was excised from anSDS-PAGE gel and electroeluted according to the manufacturer'sinstructions (BioRad). Polyclonal and monoclonal antibodies were raisedagainst ginS in rabbits and mice respectively (Institute of Infectionsand Immunity, Queens' Medical Centre, Nottingham, UK). Rabbits weresubcutaneously injected with 10-50 μg of purified protein four times, attwo weekly intervals. Serum from the rabbits was left overnight at 4° C.to clot and then centrifuged at 3,000×g for 15 min to remove remainingred blood cells (Goding, 1980). To adsorb non-specific antibodies frompolyclonal ginS serum, 200 ml of E.coli DH5α (pMalc2) was grown in LBand centrifuged at 10,000×g to harvest the cells. The cells wereresuspended in 20 ml PBS and lysed in a French press. The lysate (2 ml)was mixed with 2 ml of rabbit anti-ginS serum at room temperatureovernight. Azide (0.02%) was added to prevent contamination.

Example 9 Western Blot Analysis

[0172] Western blots were carried out, as described by Hardie, K. R. etal, 1996, except phosphate buffered saline (PBS) containing 0.5%Tween-20 was used instead of TBST, on cell lysates throughout the growthcurve of E.coli DH5α, P.gingivalis wildtype and ginS mutant. Theprotease inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK) wasadded at a concentration of 50 mM to cells of P.gingivalis prior to celllysis. Samples were boiled, resolved by SDS-PAGE and transferred tonitrocelluose. The primary antibody, ginS polyclonal serum was used at1:10,000 followed by rabbit Protein-A-alkaline phosphatase secondaryantibody at 1:1000 and the blots were developed using either AmershamECL kit or SigmaFast tablets according to the manufacturer'sinstructions.

Example 10 Generation of a P.gingivalis ginS⁻ Null Mutant

[0173] Plasmid pHGin2 was digested with EcoRI and BamHI to release theshortened version of ginS. The fragment was purifed from an agarose geland subcloned into similarly digested pUC18 (Pharmacia), to create theplasmid pHGin18. Plasmid pVA2198 was digested with EcoRI and BamnHI torelease an erythromycin cassette (erm), which was subsequently purifiedand ligated into pUC18Not (Herrero et al, 1990) at similar restrictionsites. The erm cassette was isolated from pUC18Not by digestion withNotI and subsequently cloned into the NotI site of pHGin18, thuscreating pGinerm. This construct was electroporated into P.gingivalisW50 as described by Rangarajan et al (Rangarajan, et al, Mol. Micro. 23:955-965 (1997). Transformants were selected on FAA containingclindamycin.

Example 11 N-Terminal Sequence Analysis

[0174] Cell-free culture supernatant (30 ml) from the P.gingivalis W50wildtype strain was TCA precipitated and resuspended in 400 μl of PBS.SDS sample buffer was added and 6×15 μl aliquots of protein wereresolved on a 12.5% SDS-polyacrylamide gel. The proteins were blottedonto Hybond-P PVDF membrane (Amersham Life Science) in CAPS buffer usingan XCell II unit supplied by Novex and run at 100 mA overnight. Themembrane was stained in Naphthalene black briefly, then destained indistilled water. Proteins of approximately 55 and 48 kDa were excisedfrom the membrane and protein sequencing was carried out using an ABI473A Protein Sequencer according to the manufacturer's instructions(Perkin Elmer Corp., Foster City, La., USA) by the Nottingham AutomatedSequencing Facility.

Example 12 Protease Assay

[0175] Protease activity was measured according to the method describedby Rangarajan et al (Rangarajan et al, Mol. Micro. 23: 955-965 (1997).Using an ELISA plate reader (Labsystems iEMS Reader MF) at 30° C., thereaction containing the enzyme sample in 1 ml of 0.5 mM DL-BApNA/10 mML-cysteine/10 mM CaCl₂/100 mM Tris/HCl buffer (pH 8.1) was monitored byincrease in absorbance at 405 nm due to p-nitroanilide release.

Example 13 Haemagglutination Assay

[0176] Sheep erythrocytes (SRBC) were harvested and washed twice in PBSat 4° C. by centrifugation at 2,000×g for 3 min and diluted to 0.5%(v/v) in PBS. P.gingivalis W50 wildtype and ginS mutant were grown to anA₅₈₀ of between 0.6 and 1.0, washed once in PBS and resuspended to anA₅₈₀ of 1.0. Bacterial cells (50 μl) were added to the wells ofV-shaped, 96-well plates and doubling dilutions were prepared across theplates. The SRBC (50 μl) were added and the plates were covered andincubated at 4° C. overnight. Lack of haemagglutination of SRBC wasobserved as a dark red pellet in the centre of the well. E.coli DH5αcells were used as a non-agglutination control and the P.gingivaliswildtype was a positive indicator of agglutination. The plates wereexamined and scored visually.

Example 14 P.gingivalis Does Not Possess A Gene(s) Homologous toV.fischeri LuxR/I

[0177] Many Gram-negative bacteria use homologues of the Vibrio fischeriquorum sensing luxR/I genes (Swift et al, TIBS 21: 214-219 (1996). TheLuxI proteins synthesize AHL molecules which can be detected in spentculture supernatants using a variety of biosensors. These biosensorsrespond to a particular range of AHLs depending on the length of theiracyl side chains. Spent culture supernatants from P.gingivalis W50 wereassayed for production of short and long chain AHLs using theChromobacterium mutant, CV026, and E.coli containing the luxCDABE-basedplasmid reporters, pSB401, pSB1075 and pSB1142. This was carried outusing several methods including plate assays and by overlayingThin-Layer Chromatography plates to separate AHLs and prevent inhibitionof one by the other. No production of violacin pigment or induction ofluminescence, using a Luminograph LB980 photon imaging camera, wasdetected. The unfinished P.gingivalis W83 genome sequence database(www.forsyth.org/-pggp/) was searched for homologues of LuxR/I using theNCBI Blast server and the TBLASTN program but no homologues have beenfound as yet. In parallel, the existence of functional homologues wasinvestigated by screening a genomic library against the full range ofAHL biosensors. This approach also failed to detect any AHL production.

Example 15 Identification of a luxS Homologue in P.gingivalis

[0178] In 1999, Surette et al reported that a gene, luxS, is responsiblefor production of a signalling molecule (AI-2) by Vibrio harveyi.Homologues of luxS are also found in many other bacteria includingEscherichia coli and Salmonella typhimurium (Surette et al, 1999a & b).A TBLASTN search of the P.gingivalis genome database with V.harveyi LuxSrevealed a luxS homologue, which codes for a protein of 159 amino acidsand has since been designated, ginS. The protein sequence of GinS(predicted to be 18.5 kDa) bears low amino-acid homology (30%) withother LuxS proteins from E.coli strains and Helicobacter pylori (Suretteet al, 1999a). GinS is most closely related (50% identity) to the LuxSof Borrelia burgdorferi, which causes Lyme disease (see Example 16).

Example 16 Amino-Acid Homology Between GinS and Borrelia burgorferi LuxSfrom BLAST Search

[0179] Locus: LUXS_BORBU 157 aa MAY 30, 2000

[0180] Definition: Autoinducer-2 production protein, luxS (AI-2synthesis protein).

[0181] Accession No. 050164

[0182] PID: g7387852 Version 050164 gi:7387852

[0183] Database Source: SwissProt

[0184] Source: Lyme disease spirochete, Borrelia burgorferi.

[0185] >sp|O501641|LUXS BORBU Autoinducer-2 Production Protein LuxS(AI-2 Synthesis Protein)

[0186] Length=157 aa

[0187] Score=165 bits (413), Expect=3e-40

[0188] Identities=79/158 (50%), Positives=108/158 (68%), Gaps=1/158 (0%)

[0189] Query: 1 (ginS)[SEQ ID NO:9]MEKIPSFQLDHIRLKRGIYVSRKDYIGGEVVTTFDIRMKEPNREPVLGAPELHTIEHLAA 60 M+KI SF+DH+L GIYVSRKD  +TT DIR+K PN EP++ +HTIEH AMKKITSFTIDHTKLNPGIYVSRKDTFENVIFTTIDIRIKAPNIEPIIENAAIHTIEHIGA 60 Sbjct: 1(luxS)

[0190] Query: 61 (ginS)[SEQ ID NO:10]TYLRNHPLYKDRIVFWGPMGCLTGNYFLMRGDYVSKDILPLMQETFRFIRDFEGEVPGTE 120 TLRN+++++IV++GPMGC TG Y ++GDY SKD++L+ F I+F+PGTLLRNNEVWTEKIVYFGPMGCRTGFYLIIFGDYESKDLVDLVSWLFSEIVNFSEPIPGAS 120 Sbjct:61 (luxS)

[0191] Query: 121 (ginS)[SEQ ID NO:11]PRDCGNCLLHNLPMAKYEAEKYLREVLDVATEENLNYP 158 ++CGN HNL MAKYE+KYL ++L+EENLYP DKECGNYKEHNLDMAKYESSKYL−QILNNIKENELKYP 157 Sbjct: 121 (luxS)

[0192] Sbjct: 121 (luxS)

Example 17 Complementation of AI-2 Production in E.coli DH5α

[0193] We tested whether ginS had sufficient homology to LuXSEC tocomplement the mutation in DH50α. GinS was amplified from P.gingivalisW50 genomic DNA and cloned into pHG327 to create pGin1. SDS-PAGEanalysis of whole cell extracts of E.coli DH5α (pHGin1) failed to revealthe production of a protein at the predicted MW for GinS (18.5 kDa).However, production of AI-2 was detected in spent culture supernatantusing the V.harveyi BB170 (see FIGS. 1 and 2). Consequently, ginS wascloned into pMal-c2 (pMalGin 1) and MalE-GinS (ca. 62 kDa) was producedin DH5α Analysis of spent culture supernatant of this strain using thesame V.harveyi BB170 sensor, likewise demonstrated functionalcomplementation of the LuxS_(Ec) mutation in DH5α by production of theAI-2 molecule (see FIG. 4). The functionally active MalE-GinS wassubsequently purified by affinity chromatography (FIG. 5). Cleavage ofGinS from MalE was successfully performed using Factor Xa as describedin materials and methods (see FIG. 6). This purified GinS was used toraise specific polyclonal antisera to GinS in rabbits. Western blottingof whole cell extracts of E.coli DH5α (pMalGin1) and analysis of spentculture supernatant from E.coli DH5α (pMalGin1) supernatants attime-points throughout the growth curve, showed that AI-2 productioncommences within 4 of growth (mid-exponential phase) and continuesthrough stationary phase, with some degradation evident by 9 hours (seeFIG. 8).

Confirmation of GinS Expression and AI-2 Production in E.coli andP.gingivalis.

[0194]P.gingivalis W50 wildtype spent BHI culture supernatant, taken atvarious points throughout the growth curve, was assayed for productionof AI-2 against V.harveyi BB170, but no activity was detected. However,when grown in a defined medium, the molecule was maximally detected atmid-expotential phase of growth whilst Western blots revealed that inE.coli, GinS accumulates throughout growth and is still increasing at 24hours (see FIG. 8). In P.gingivalis, GinS is detected first at 6 hoursand appears to still be increasing at 96 hours (see FIG. 9). A ginS⁻null mutant of P.gingivalis was prepared as described in materials andmethods and confirmed by PCR by the presence of a larger amplified DNAfragment corresponding to insertion of the erm cassette (FIG. 10).Southern blot analysis confirmed a single erm insertion in thechromosome (FIG. 11). Genomic DNA was digested by BalI which cuts oncewithin ginS. This site is removed by the mutation. The digested DNA wasprobed with a DIG-labelled ginS PCR fragment. Wildtype genomic DNAshowed two fragments hybridised to the probe as expected, whereas withthe mutant DNA, a single, larger fragment was detected showing that theerm cassette had replaced the BalI restriction site. Amplification andcloning of the 5′ and 3′ regions of the ginS⁻ mutant, followed bysequencing of the clones, further confirmed that the ginS mutant hadinserted into the correct region of the chromosome. The absence of GinSin Western blots of the ginS⁻ mutant further confirmed deletion of thegene (FIG. 9, Panel B).

GinS Regulates Expression and Secretion of Rgp Proteases (Gingipains),as well as Haemagglutination of Erythrocytes in P.gingivalis

[0195] The major virulence factors produced by P.gingivalis are thesecreted proteases or gingipains, Rgp and Kgp. To discover whether GinSis involved in protease regulation, the protease profile and activity ofthe wildtype and ginS⁻ null mutant of P.gingivalis was compared bySDS-PAGE and protease assays using BApNA and Z-Lys-pNA. SDS-PAGEanalysis of TCA precipitated spent culture supernatants of the wildtypeand ginS⁻ mutant revealed a down-regulation of two proteins (55 and 48kDa) in the ginS⁻ null mutant (see FIG. 13).

[0196] N-terminal sequencing of these proteins from the wildtyperevealed sequences of DVYTDHGDLYNT [SEQ ID NO:12] and YTPVEEKQNGRM [SEQID NO:13] respectively, identifying the proteins as Rgp and Kgp. BApNAand Z-Lys-pNA specific assays of total cultures, cell-free supernatantsand lysates revealed a significant decrease in protease activity of theginS⁻ mutant when compared with the wildtype (see FIG. 14)

[0197] Initial analysis of KGP and RGP activity in the soluble cellularfractions demonstrated an accumulation in the mutant vs. the wildtype,suggesting that overall expression of proteases are unaffected by theinsertional mutation in ginS⁻, but rather these data strongly suggest arole for GinS the regulation of protease export/secretion from the cell.However, the more detailed data obtained via analysis of the enzymaticactivity in whole cell lysates (see FIG. 14 (Table 3)) analysis clearlyindicates that in addition to a possible influence on proteasesecretion/export, RgpB expression itself and possibly also RgpA, aredown-regulated in the ginS⁻ null mutant, suggesting that ginS⁻ isdirectly involved in regulating gene expression of an importantvirulence determinants directly involved in host tissue destruction. 2-Dgel electrophoresis was carried out according to a standard protocol(see Berkelman, et al., 2-D Electrophoresis: Principles and Methods.Amersham Pharmacia Biotech(ftp://ftp.hpb.com/pdf/2D_Brochure_(—)13.pdf), in order to identifydifferences in the protein expression patterns between wildtypeP.gingivalis and the ginS⁻ null mutant which might indicate regulatorycontrol by AI-2, produced as a result of ginS expression. A single spot,designated WT2, was identified as being expressed exclusively in thewildtype and was excised and subjected to further analysis andcharacterization in order to obtain an identification (see Examples 30and 31).

[0198] Methods: Sample Preparation for Mass Spectrometry

[0199] In-gel digestion of the proteins was performed on a automateddigester (DigestPro™, AbiMed, Germany) using a standard protocol. Afterextraction of peptides from the gel, the solution was dried in a vacuumcentrifuge. The peptides were resuspended in 10 μl 5% formic acid. ForNanospray MS analysis, five microliter of this solution were purifiedusing a 10 μl pipette tip loaded with a C18 resin (ZipTip, Millipore).The bed volume of the resin of 0.6 μl was reduced by approximately halfby cutting of a small piece form the front of the tip. The pipette tipwas wetted with water/acetonitrile (1:2) and equilibrated with aqueous0.2% trifluoroacetic acid. The peptides were loaded by repeatedlyaspirating and dispensing the 5 μl peptide solution. The tip was washedby aspirating and dispensing of 5% formic acid for four to five times.Peptides were eluted in 1-2 μl of water/methanol (1:1) containing 5%formic acid. The eluate was immediately loaded into a nanospraymicrocapillary (type “N”, Protana, Odense, Denmark). After opening ofthe tip by touching a glass slide under a stereomicroscope, themicrocapillary was placed in the ion source of a Qtof mass spectrometerfor analysis.

[0200] Mass spectrometry. MALDI Tof MS was performed on a TofSpec SEinstrument from Micromass, Manchester, UK. Nanospray MS and MS/MSexperiments were performed on a orthogonal accelerationquadrupole-time-of-flight mass spectrometer (Q-Tof, Micromass,Manchester, UK) equipped with a Z-spray ion source for Nanosprayanalysis.

[0201] Database Searches

[0202] Peptide mass searches and sequence tag searched were performedusing PepSea™ software (Protana, Denmark) that was installed in-houseagainst a upto date non-redundant protein database that was downloadedfrom the National Center for Biotechnology Information (NCBI) andagainst a database containing bacterial proteins.

[0203] The protein in sample WT2 was putatively identified as PG33, animmunoreactive 42 kDa antigen of P.gingivalis (NCBI accession no.AF175715.1 ;gi:5759279, deposited by Ross et al, 1998 and 1999; seeExamples 32 and 33).

Example 18 Search Results Sample WT2 Search (Auto 16) (WT2) Second ass

[0204] Entry name: Wild-type P.gingivalis strain W50, spot 2.

[0205] Average mass, pI: 43117.161 [Da,unmodified], 8.35.

[0206] DB Accession no.: gi:5759279.

[0207] Score: 237.

[0208] Matches: 10/55.

[0209] Total coverage; 123/385 (31.95%).

[0210] 2RMS: 0.030 [Da] 20.920 [ppm].

[0211] Match Results

[0212] Masses are Protonated (MH+).

[0213] Cysteine is Carbamidomethyl-Cys. Methionine is Native MeasuredCalculated Mono [Da] [Da] [Da] [ppm] Diff. Diff. Start End Sequence1131.674 1131.677 Yes −0.003 −2.850 75 85 (R)LSIVPTFGIGK(W) [SEQ IDNO:14] 1192.589 1192.553 Yes 0.036 30.196 86 94 (K)WHEPYFGTR(L) [SEQ IDNO:15] 1209.701 1209.663 Yes 0.038 31.658 280 289 (R)VVVDNVVYFR(J) [SEQID NO:16] 1493.719 1493.734 Yes −0.015 −9.866 169 182(K)DDMTGTVNVGLMLK(F) [SEQ ID NO:17] 1656.729 1656.786 Yes −0.057 −34.723298 311 (R)NQEINVYNTAEYAK(T) [SEQ ID NO:18] 1682.725 1682.741 Yes −0.016−9.219 364 377 (K)GSSEQIYEENAWNR(I) [SEQ ID NO:19] 1804.854 1804.890 Yes−0.036 −20.158 95 110 (R)LQETGFDIYGFPQGSK(E) [SEQ ID NO:20 2040.9702040.999 Yes −0.028 −13.878 295 311 (K)IDRNQEINVYNTAEYAK(T) [SEQ IDNO:21 2110.969 2111.001 Yes −0.032 −15.326 262 279(R)RPVSCPECPEPTQPTVTR(V) [SEQ ID NO:22]

Cysteine is Carbamidomethyl-Cys. Methionine is Oxidized

[0214] Measured Calculated Mono [Da] [Da] [Da] [ppm] Diff. Diff. StartEnd Sequence 1613.793 1613.806 Yes −0.014 −8.505 327 340(K)TGTAAYNMKLSERR(A) [SEQ ID NO:23]

Cysteine is Acrylamido-Cys. Methionine is Native

[0215] No peptides matched.

Cysteine is Acrylamido-Cys. Methionine is Oxidized

[0216] No peptides matched.

Example 19 Amino Acid Sequence of Protein Identified as Highest Matchwith Peptides Obtained by Digestion of Excised Spot WT2.

[0217] MTYRIMKAKS LLLALAGLAC TFSATAQEAT TQNKAGMHTA FQRDKASDHW FTDIAGGAGMALSGWNNDVD FVDR LSIVPT FGIGKWHEPY FGTRLOFTGF DIYGFPQGSK ERNHNYFGNAHLDFMFDLTN YFGVYRPNRV FHIIPWAGIG FGYKFHSENA NGEKVGSK DD MTGTVNVGLM LKFRLSRVVD FNIEGQAFAG KMNFIGTKRG KADFPVMATA GLTFNLCKTE WTEIVPMDYALVNDLNNQIN SLRGQVEELS R RPVSCPECP EPTQPTVTRV VVDNVVYFRI NSAKIDRNQE INVYNTAEYA K TNNAPIKVV GYADEK TGTA AYNMKLSERR  AKAVAKMLEKYGVSADRITI EWK GSSEQIY EENAWNR IVV MTAAE

[0218] n.b. Peptides obtained by digestion of spot WT2 are underlined

Example 20 Nucleotide Sequence of Porphyromonas gingivalis Strain W50Immunoreactive 42kD Antigen PG33 Gene (Accession no. AF175715.1;gi:5759278)

[0219] [SEQ ID NO:25] 5′-ATCAAAGCTAAATCTTTATTATTAGCACTTGCGGGTCTCGCATGCACATTCAGTGCAACAGCCCAAGAAGCTACTACACAGAACAAGCAGGGATGCACACCGCATTCCAACGTGATAAGGCCTCCGATCATTGGTTCATTGACATTGCAGGTGGAGCAGGTATGGCTCTCTCGGGATGGAATSSTGATGTAGACTTTGTAGATCGTCTAAGTATCGTTCCTACTTTCGGTATCGGTAAATGGCATGAGCCTTATTTCGGTACTCGTCTCCAATTCACAGGATTCGACATCTATGGATTCCCGCAAGGGAGCAAGGAGCGTAACCACAATTACTTTGGAAACGCCCACCTTGACTTCATGTTCGATCTGACGAACTATTTCGGTGTATACCGTCCCAATCGTGTCTTCCATATCATCCCATGGGCAGGTATAGGATTTGGTTATAAATTCCATAGCGAAAACGCCAATGGTGAAAAAGTAGGAAGTAAAGATGATATGACCGGAACAGTTAATGTCGGTTTGATGCTGAAATTCCGCCTATCAAGAGTCGTAGACTTCAATATTGAAGGACAAGCTTTTGCCGGAAAGATGAACTTTATCGGGACAAAGAGAGGAAAAGCAGACTTCCCTGTAATGGCTACAGCAGGTCTAACGTTCAACCTTGGCAAGACAGAGTGGACAGAAATTGTTCCTATGGACTATGCTTTGGTCAATGACCTGAACAACCAAATCAACTCACTTCGCCGTCAAGTGGAAGAGTTGAGCCGTCGTCCTGTTTCATGCCCTGAATGCCCTGAGCCTACACAGCCTACAGTTACTCGTGTAGTCGTTGACAATGTGGTTTACTTCCGTATCAATAGTGCAAAGATTGATCGTAATCAAGAAATCAATGTTTACAATACAGCTGAATATGCGAAGACCAACAACGCACCGATCAAGGTAGTAGGTTACGCTGACGAAAAAACCGGTACTGCGGCCTATAACATGAAGCTTTCAGAGCGTCGTGCAAAAGCGGTAGCCAAGATGCTTGAAAAGTATGGTGTTTCTGCGGATCGCATTACAATTGAATGGAAGGGCTCATCAGAGCAAATCTATGAAGAGAACGCTTGGAATCGTATTGTAGTAATGACTGCAGCGGAATAA-3′

Example 21 Amino Acid Sequence of Porphyromonas gingivalis strain W50immunoreactive 42 kD antigen PG33 gene (Accession no. AF175715.1;gi:5759278)

[0220] [SEQ ID NO:26] NH₂-MKAKSLLLALAGLACTFSATAQEATTQNKAGMHTAFQRDKASDHWFIDIAGGAGMALSGWNNDVDFVDRLSIVPTFGIGKWHEPYFGTRLQFTGFDIYGFPQGSKERNHNYFGNAHLDFMFDLTNYFGVYRPNRVFHIIPWAGIGFGYKFHSENANGEKVGSKDDMTGTVNCGLMLKFRLSRVVDFNIEGQAFAGKMNFIGTKRGKADFPVMATAGLTFNLGKTEWTEIVPMDYALVNDLNNQINSLRGQVEELSRRPVSCPECPEPTQPTVTRVVVDNVVYFRINSAKIDRNQEINVYNTAEYAKTNNAPIKVVGYADEKTGTAAYNMKLSERRAKAVAKMLEKYGVSADRITIEWKGSSEQIYEENAWNRIVVMTAAE-COOH

Example 22 Alignment (Clustal Method) of the Amino Acid Sequences ofSpot WT2/ sb/pog|344208 and the P.gingivalis P33 42 kDa antigen (NCBIAF175715.1;gi:5755279) Using the Megalign™ Algorithm, DNASTAR, Inc.,Showing 100% Identity

[0221] M T Y R I M K A K S L L L A L A G L A C T F S A T A Q E A TMajority {overscore(                  |                   |                   | )}                  10                  20                  30                  |                   |                   |  1 M T Y R IM K A K S L L L A L A G L A C T F S A T A Q E A T WT2 Spot.pro 1M - - - - - K A K S L L L A L A G L A C T F S A T A Q E A T p33 Ag.pro TQ N K A G M H T A F Q R D K A S D H W F I D I A G G A G M Majority{overscore(                  |                   |                   | )}                  40                  50                  60                  |                   |                   |  31 T Q N KA G M H T A F Q R D K A S D H W F I D I A G G A G M WT2 Spot.pro 26 T QN K A G M H T A F Q R D K A S D H W F I D I A G G A G M p33 Ag.pro A L SG W N N D V D F V D R L S I V P T F G I G K W H E P Y Majority{overscore(                  |                   |                   | )}                  70                  80                  90                  |                   |                   |  61 A L S GW N N D V D F V D R L S I V P T F G I G K W H E P Y WT2 Spot.pro 56 A LS G W N N D V D F V D R L S I V P T F G I G K W H E P Y ×Ag.pro F G T RL Q F T G F D I Y G F P Q G S K E R N H N Y F G N A Majority {overscore(                  |                   |                   | )}                 100                 110                 120                  |                   |                   |  91 F G T RL Q F T G F D I Y G F P Q G S K E R N H N Y F G N A WT2 Spot.pro 86 F GT R L Q F T G F D I Y G F P Q G S K E R N H N Y F G N A p33 Ag.pro H L DF M F D L T N Y F G V Y R P N R V F H I I P W A G I G Majority{overscore(                  |                   |                   | )}                 130                 140                 150                  |                   |                   |  121 H L D FM F D L T N Y F G V Y R P N R V F H I I P W A G I G WT2 Spot.pro 116 H LD F M F D L T N Y F G V Y K P N R V F H I I P W A G I G p33 Ag.pro F G YK F H S E N A N G E K V G S K D D M T G T V N V G L M Majority{overscore(                  |                   |                   | )}                 160                 170                 160                  |                   |                   |  151 F G Y KF H S E N A N G E K V G S K D D M T G T V N V G L M WT2 Spot.pro 146 F GY K F H S E N A N G E K V G S K D D M T G T V N V G L M p33 Ag.pro L K FR L S R V V D F N I E G Q A F A G K M N F I G T K R G Majority{overscore(                  |                   |                   | )}                 190                 200                 210                  |                   |                   |  181 L K F RL S R V V D F N I E G Q A F A G K M N F I G T K R G WT2 Spot.pro 176 L KF R L S R V V D F N I E G Q A F A G K M N F I C T K R g p33 Ag.pro K A DF P V M A T A G L T F N L G K T E W T E I V P M D T A Majority{overscore(                  |                   |                   | )}                 220                 230                 240                  |                   |                   |  211 K A D FP V M A T A G L T F N L G K T E W T E I V P M D Y A WT2 Spot.pro 206 K AD F P V M A T A G L T F N L G K T E W T E I V P M D Y A p33 Ag.pro L V ND L N N Q I N S L R G V E E L S R R P V S C P E C P Majority {overscore(                  |                   |                   | )}                 250                 260                 270                  |                   |                   |  241 L V N DL N N Q I N S L R G Q V E E L S R R P V S C P E C P WT2 Spot.pro 236 L VN D L N N Q I N S L R G Q V E E L S R K P V S C P E C P p33 Ag.pro E P TQ P T V T R V V V D N V V YV F R I N S A K I D R N Q E Majority{overscore(                  |                   |                   | )}                 280                 290                 300                  |                   |                   |  271 E P T QP T V T R V V V D N V V Y F R I N S A K I D R N Q E WT2 Spot.pro 266 E PT Q P T V T K V V V D N V V Y F R I N S A K I D R N Q E p33 Ag.pro I N VY N T A E Y A K T N N A P I K V V G Y A D E K T G T A Majority{overscore(                  |                   |                   | )}                 310                 320                 330                  |                   |                   |  301 I N V YN T A E Y A K T N N A P I K V V G Y A D E K T G T A WT2 Spot.pro 296 I NV Y N T A E Y A K T N N A P I K V V G Y A D E K T G T A p33 Ag.pro A Y NM K L S E R R A K A V A K M L E K Y G V S A D R I T I Majority{overscore(                  |                   |                   | )}                 340                 350                 360                  |                   |                   |  331 A Y N MK L S E R R A K A V A K M L E K Y G V S A D R I T I WT2 Spot.pro 326 A YN M K L S E R R A K A V A K M L E K Y G V S A D R I T I p33 Ag pro E W KG S S E Q I Y E E N A W N R I V V M T A A E Majority {overscore(                  |                   |)}                 370                 380                  |                   | 361 E W K G S S E Q I Y E E N AW N R I V V M T A A E WT2 Spot.pro 356 E W K G S S E Q I Y E E N A W N RI V V M T A A E p33 Ag.pro

[0222] Discussion of Previously Described Data (see Examples 1-22 andFIGS. 1-19)

[0223] Quorum sensing describes a bacterial signalling mechanism,whereby, accumulation of molecules, known as autoinducers, allowsindividual bacteria to sense their environment and respond by regulatinggene expression. Many Gram-negative bacteria employ a range ofN-acyl-L-homoserine lactone (AHL) molecules as their signals andregulate expression of phenotypes, such as bioluminescence, usinghomologues of the V.fischeri LuxR/I proteins (Williams et al, FEMSMicro. Lett. 100: 161-168 (1992). However, a number of bacteria,including E.coli, B.subtilis and H.pylori, produce a molecule known asAI-2, the synthesis of which is driven by a homologue of the VharveyiLuxS protein (Surette, et al., Proc. Natl. Acad. Sci, USA. 96: 1639-1644(1999a). Forsyth, et al., Infect. Imman. 68: 3193-3199 (2000); Joyce etal, J. Bacteriol. 182: 3638-3643 (2000). AI-2 appears to represent a newfamily of signal molecules and it has been suggested that it is involvedin cross-communication of bacteria because of its presence in bothGram-negatives and Gram-positives (Bassler et al,. J. Bacteriol. 179:4043-4045 (1997)). In this study we have shown that P.gingivalispossesses a functional LuxS homologue, which we have designated GinS.This is the first evidence of a quorum sensing system in an anaerobichuman pathogen. LuxS controls bioluminescence in V.harveyi (Surette etal, Proc. Natl. Acad. Sci, USA. 96: 1639-1644 (1999a)) and type IIIsecretion in enterohemorrhagic (EHEC) and enteropathogenic (EPEC)E.coli, (Sperandio et al, PNAS 96: 15196-15201 (1999). A P.gingivalisginS⁻ null mutant shows down-regulation of the major extracellularcysteine proteases, Rgp and Kgp, in culture supernatant in addition to a4-fold decrease in haemagglutination of erythrocytes. These findingssuggest that GinS plays a major role in the control of these importantvirulence factors. This finding is consistent with previous reports thatcysteine proteases and haemagglutinin activities of P.gingivalis appearto be structurally related (Pavloff, et al., J. Biol. Chem. 270:1007-1010 (1995).; Yoneda, et al., Genetic evidence for the relationshipof Porphyromonas gingivalis cysteine protease and hemagglutininactivities. Oral Microbiol Immunol. 11: 129-134 (1996).

[0224] GinS bears low amino-acid homology with LuxS in E.coli. However,overproduction of the protein in E.coli DH50α and subsequent screeningspent culture supernatant against V.harveyi sensor BB 170, complementedAI-2 production. This verifies that ginS encodes a related molecule tothat of the other LuxS homologues. Detection of the signal molecule inP.gingivalis was achieved by growing the bacterium in a chemicallydefined medium and screening spent supernatants against BB170. Themolecule is produced at mid-exponential growth and depleted by earlystationary phase, which is consistant with results obtained from E.coliharbouring GinS. Western blot analysis of production of GinS inP.gingivalis and E.coli throughout the growth curve showed that theprotein persists into late stationary phase, whereas the autoinducerdepletes. It is predicted in E.coli and S.typhimitrium that there is anAI-2 degradation pathway (Surette and Bassler, 1998; Surette et al,1999), therefore, it is interesting that the protein is not similarlylost. This perhaps suggests that the protein persists for directactivity under certain conditions and may differ in vivo, or it may havea secondary role as yet uncharacterised.

[0225] Using established biosensors and construction and analysis of aP.gingivalis W50 chromosomal library, no AHL molecules or LuxR/Ihomologues were identified. In addition, a search of the P.gingivalisgenome sequence database provided further evidence that a V.fischerisystem is not employed by this bacterium. However, because the genomesequence is still unfinished, these results are inconclusive.

[0226] We predict that GinS is active in P.gingivalis because of theregulation of protease expression and activity, as well ashaemagglutinin activity, together with the ability of spent culturesupernatants from P.gingivalis to induce luminescence in V.harveyi BB170. Further work will involve determining if the molecule can restoreprotease and haemagglutinin activity in the ginS⁻ null mutant and usinga number of techniques such as 2-D gel electrophoresis in attempt toisolate other associated targets.

[0227] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1 26 1 480 DNA Porphyromonas gingivalis 1 atggaaaaaa ttcccagttttcagttagat catattcgcc tcaaacgagg catatatgtc 60 tcccgcaagg actatatagggggagaggtg gttacgactt tcgatattcg aatgaaagag 120 cccaatcgcg aaccggtgcttggggcaccc gaactgcata cgatcgagca tttggctgca 180 acttatctgc gtaatcatccgctttataag gacaggatcg ttttctgggg gccgatgggc 240 tgccttacgg gcaattactttctgatgcga ggcgattacg tatccaaaga tatactgccc 300 ctcatgcagg agactttccgcttcatcaga gacttcgaag gagaagtgcc gggtacggag 360 ccgcgcgact gtggcaactgcctgctgcac aacctgccga tggccaaata tgaggccgag 420 aaatacctgc gtgaggtactcgatgtagcg acggaggaga acctgaacta tcccgactga 480 2 159 PRT Porphyromonasgingivalis 2 Met Glu Lys Ile Pro Ser Phe Gln Leu Asp His Ile Arg Leu LysArg 1 5 10 15 Gly Ile Tyr Val Ser Arg Lys Asp Tyr Ile Gly Gly Glu ValVal Thr 20 25 30 Thr Phe Asp Ile Arg Met Lys Glu Pro Asn Arg Glu Pro ValLeu Gly 35 40 45 Ala Pro Glu Leu His Thr Ile Glu His Leu Ala Ala Thr TyrLeu Arg 50 55 60 Asn His Pro Leu Tyr Lys Asp Arg Ile Val Phe Trp Gly ProMet Gly 65 70 75 80 Cys Leu Thr Gly Asn Tyr Phe Leu Met Arg Gly Asp TyrVal Ser Lys 85 90 95 Asp Ile Leu Pro Leu Met Gln Glu Thr Phe Arg Phe IleArg Asp Phe 100 105 110 Glu Gly Glu Val Pro Gly Thr Glu Pro Arg Asp CysGly Asn Cys Leu 115 120 125 Leu His Asn Leu Pro Met Ala Lys Tyr Glu AlaGlu Lys Tyr Leu Arg 130 135 140 Glu Val Leu Asp Val Ala Thr Glu Glu AsnLeu Asn Tyr Pro Asp 145 150 155 3 31 DNA Porphyromonas gingivalis 3gtattatcag cggaattccc ggcgaaggtc g 31 4 32 DNA Porphyromonas gingivalis4 gataccgcct ccggatccaa taatccatcc gg 32 5 33 DNA Escherichia coli 5gcggccgcca ccaaatgctc gatcgtatgc cag 33 6 31 DNA Escherichia coli 6tggcggccgc gcgtgaggta ctcgatgtag g 31 7 25 DNA Porphyromonas gingivalis7 agacaatccc gaattcgaga tggaa 25 8 24 DNA Porphyromonas gingivalis 8tgagaaatag agcggatcct aagc 24 9 60 PRT Borrelia burgorferi 9 Met Glu LysIle Pro Ser Phe Gln Leu Asp His Ile Arg Leu Lys Arg 1 5 10 15 Gly IleTyr Val Ser Arg Lys Asp Tyr Ile Gly Gly Glu Val Val Thr 20 25 30 Thr PheAsp Ile Arg Met Lys Glu Pro Asn Arg Glu Pro Val Leu Gly 35 40 45 Ala ProGlu Leu His Thr Ile Glu His Leu Ala Ala 50 55 60 10 60 PRT Borreliaburgorferi 10 Thr Tyr Leu Arg Asn His Pro Leu Tyr Lys Asp Arg Ile ValPhe Trp 1 5 10 15 Gly Pro Met Gly Cys Leu Thr Gly Asn Tyr Phe Leu MetArg Gly Asp 20 25 30 Tyr Val Ser Lys Asp Ile Leu Pro Leu Met Gln Glu ThrPhe Arg Phe 35 40 45 Ile Arg Asp Phe Glu Gly Glu Val Pro Gly Thr Glu 5055 60 11 38 PRT Borrelia burgorferi 11 Pro Arg Asp Cys Gly Asn Cys LeuLeu His Asn Leu Pro Met Ala Lys 1 5 10 15 Tyr Glu Ala Glu Lys Tyr LeuArg Glu Val Leu Asp Val Ala Thr Glu 20 25 30 Glu Asn Leu Asn Tyr Pro 3512 12 PRT Porphyromonas gingivalis 12 Asp Val Tyr Thr Asp His Gly AspLeu Tyr Asn Thr 1 5 10 13 12 PRT Porphyromonas gingivalis 13 Tyr Thr ProVal Glu Glu Lys Gln Asn Gly Arg Met 1 5 10 14 13 PRT Porphyromonasgingivalis 14 Arg Leu Ser Ile Val Pro Thr Phe Gly Ile Gly Lys Trp 1 5 1015 11 PRT Porphyromonas gingivalis 15 Lys Trp His Glu Pro Tyr Phe GlyThr Arg Leu 1 5 10 16 12 PRT Porphyromonas gingivalis 16 Arg Val Val ValAsp Asn Val Val Tyr Phe Arg Ile 1 5 10 17 16 PRT Porphyromonasgingivalis 17 Lys Asp Asp Met Thr Gly Thr Val Asn Val Gly Leu Met LeuLys Phe 1 5 10 15 18 16 PRT Prophyromonas gingivalis 18 Arg Asn Gln GluIle Asn Val Tyr Asn Thr Ala Glu Tyr Ala Lys Thr 1 5 10 15 19 16 PRTProphyromonas gingivalis 19 Lys Gly Ser Ser Glu Gln Ile Tyr Glu Glu AsnAla Trp Asn Arg Ile 1 5 10 15 20 18 PRT Porphyromonas gingivalis 20 ArgLeu Gln Phe Thr Gly Phe Asp Ile Tyr Gly Phe Pro Gln Gly Ser 1 5 10 15Lys Glu 21 19 PRT Porphyromonas gingivalis 21 Lys Ile Asp Arg Asn GlnGlu Ile Asn Val Tyr Asn Thr Ala Glu Tyr 1 5 10 15 Ala Lys Thr 22 20 PRTPorphyromonas gingivalis 22 Arg Arg Pro Val Ser Cys Pro Glu Cys Pro GluPro Thr Gln Pro Thr 1 5 10 15 Val Thr Arg Val 20 23 16 PRT Porphyromonasgingivalis 23 Lys Thr Gly Thr Ala Ala Tyr Asn Met Lys Leu Ser Glu ArgArg Ala 1 5 10 15 24 385 PRT Porphyromonas gingivalis 24 Met Thr Tyr ArgIle Met Lys Ala Lys Ser Leu Leu Leu Ala Leu Ala 1 5 10 15 Gly Leu AlaCys Thr Phe Ser Ala Thr Ala Gln Glu Ala Thr Thr Gln 20 25 30 Asn Lys AlaGly Met His Thr Ala Phe Gln Arg Asp Lys Ala Ser Asp 35 40 45 His Trp PheIle Asp Ile Ala Gly Gly Ala Gly Met Ala Leu Ser Gly 50 55 60 Trp Asn AsnAsp Val Asp Phe Val Asp Arg Leu Ser Ile Val Pro Thr 65 70 75 80 Phe GlyIle Gly Lys Trp His Glu Pro Tyr Phe Gly Thr Arg Leu Gln 85 90 95 Phe ThrGly Phe Asp Ile Tyr Gly Phe Pro Gln Gly Ser Lys Glu Arg 100 105 110 AsnHis Asn Tyr Phe Gly Asn Ala His Leu Asp Phe Met Phe Asp Leu 115 120 125Thr Asn Tyr Phe Gly Val Tyr Arg Pro Asn Arg Val Phe His Ile Ile 130 135140 Pro Trp Ala Gly Ile Gly Phe Gly Tyr Lys Phe His Ser Glu Asn Ala 145150 155 160 Asn Gly Glu Lys Val Gly Ser Lys Asp Asp Met Thr Gly Thr ValAsn 165 170 175 Val Gly Leu Met Leu Lys Phe Arg Leu Ser Arg Val Val AspPhe Asn 180 185 190 Ile Glu Gly Gln Ala Phe Ala Gly Lys Met Asn Phe IleGly Thr Lys 195 200 205 Arg Gly Lys Ala Asp Phe Pro Val Met Ala Thr AlaGly Leu Thr Phe 210 215 220 Asn Leu Gly Lys Thr Glu Trp Thr Glu Ile ValPro Met Asp Tyr Ala 225 230 235 240 Leu Val Asn Asp Leu Asn Asn Gln IleAsn Ser Leu Arg Gly Gln Val 245 250 255 Glu Glu Leu Ser Arg Arg Pro ValSer Cys Pro Glu Cys Pro Glu Pro 260 265 270 Thr Gln Pro Thr Val Thr ArgVal Val Val Asp Asn Val Val Tyr Phe 275 280 285 Arg Ile Asn Ser Ala LysIle Asp Arg Asn Gln Glu Ile Asn Val Tyr 290 295 300 Asn Thr Ala Glu TyrAla Lys Thr Asn Asn Ala Pro Ile Lys Val Val 305 310 315 320 Gly Tyr AlaAsp Glu Lys Thr Gly Thr Ala Ala Tyr Asn Met Lys Leu 325 330 335 Ser GluArg Arg Ala Lys Ala Val Ala Lys Met Leu Glu Lys Tyr Gly 340 345 350 ValSer Ala Asp Arg Ile Thr Ile Glu Trp Lys Gly Ser Ser Glu Gln 355 360 365Ile Tyr Glu Glu Asn Ala Trp Asn Arg Ile Val Val Met Thr Ala Ala 370 375380 Glu 385 25 1143 DNA Porphyromonas gingivalis 25 atgaaagctaaatctttatt attagcactt gcgggtctcg catgcacatt cagtgcaaca 60 gcccaagaagctactacaca gaacaaagca gggatgcaca ccgcattcca acgtgataag 120 gcctccgatcattggttcat tgacattgca ggtggagcag gtatggctct ctcgggatgg 180 aataatgatgtagactttgt agatcgtcta agtatcgttc ctactttcgg tatcggtaaa 240 tggcatgagccttatttcgg tactcgtctc caattcacag gattcgacat ctatggattc 300 ccgcaagggagcaaggagcg taaccacaat tactttggaa acgcccacct tgacttcatg 360 ttcgatctgacgaactattt cggtgtatac cgtcccaatc gtgtcttcca tatcatccca 420 tgggcaggtataggatttgg ttataaattc catagcgaaa acgccaatgg tgaaaaagta 480 ggaagtaaagatgatatgac cggaacagtt aatgtcggtt tgatgctgaa attccgccta 540 tcaagagtcgtagacttcaa tattgaagga caagcttttg ccggaaagat gaactttatc 600 gggacaaagagaggaaaagc agacttccct gtaatggcta cagcaggtct aacgttcaac 660 cttggcaagacagagtggac agaaattgtt cctatggact atgctttggt caatgacctg 720 aacaaccaaatcaactcact tcgcggtcaa gtggaagagt tgagccgtcg tcctgtttca 780 tgccctgaatgccctgagcc tacacagcct acagttactc gtgtagtcgt tgacaatgtg 840 gtttacttccgtatcaatag tgcaaagatt gatcgtaatc aagaaatcaa tgtttacaat 900 acagctgaatatgcgaagac caacaacgca ccgatcaagg tagtaggtta cgctgacgaa 960 aaaaccggtactgcggccta taacatgaag ctttcagagc gtcgtgcaaa agcggtagcc 1020 aagatgcttgaaaagtatgg tgtttctgcg gatcgcatta caattgaatg gaagggctca 1080 tcagagcaaatctatgaaga gaacgcttgg aatcgtattg tagtaatgac tgcagcggaa 1140 taa 1143 26380 PRT Porphyromonas gingivalis 26 Met Lys Ala Lys Ser Leu Leu Leu AlaLeu Ala Gly Leu Ala Cys Thr 1 5 10 15 Phe Ser Ala Thr Ala Gln Glu AlaThr Thr Gln Asn Lys Ala Gly Met 20 25 30 His Thr Ala Phe Gln Arg Asp LysAla Ser Asp His Trp Phe Ile Asp 35 40 45 Ile Ala Gly Gly Ala Gly Met AlaLeu Ser Gly Trp Asn Asn Asp Val 50 55 60 Asp Phe Val Asp Arg Leu Ser IleVal Pro Thr Phe Gly Ile Gly Lys 65 70 75 80 Trp His Glu Pro Tyr Phe GlyThr Arg Leu Gln Phe Thr Gly Phe Asp 85 90 95 Ile Tyr Gly Phe Pro Gln GlySer Lys Glu Arg Asn His Asn Tyr Phe 100 105 110 Gly Asn Ala His Leu AspPhe Met Phe Asp Leu Thr Asn Tyr Phe Gly 115 120 125 Val Tyr Arg Pro AsnArg Val Phe His Ile Ile Pro Trp Ala Gly Ile 130 135 140 Gly Phe Gly TyrLys Phe His Ser Glu Asn Ala Asn Gly Glu Lys Val 145 150 155 160 Gly SerLys Asp Asp Met Thr Gly Thr Val Asn Val Gly Leu Met Leu 165 170 175 LysPhe Arg Leu Ser Arg Val Val Asp Phe Asn Ile Glu Gly Gln Ala 180 185 190Phe Ala Gly Lys Met Asn Phe Ile Gly Thr Lys Arg Gly Lys Ala Asp 195 200205 Phe Pro Val Met Ala Thr Ala Gly Leu Thr Phe Asn Leu Gly Lys Thr 210215 220 Glu Trp Thr Glu Ile Val Pro Met Asp Tyr Ala Leu Val Asn Asp Leu225 230 235 240 Asn Asn Gln Ile Asn Ser Leu Arg Gly Gln Val Glu Glu LeuSer Arg 245 250 255 Arg Pro Val Ser Cys Pro Glu Cys Pro Glu Pro Thr GlnPro Thr Val 260 265 270 Thr Arg Val Val Val Asp Asn Val Val Tyr Phe ArgIle Asn Ser Ala 275 280 285 Lys Ile Asp Arg Asn Gln Glu Ile Asn Val TyrAsn Thr Ala Glu Tyr 290 295 300 Ala Lys Thr Asn Asn Ala Pro Ile Lys ValVal Gly Tyr Ala Asp Glu 305 310 315 320 Lys Thr Gly Thr Ala Ala Tyr AsnMet Lys Leu Ser Glu Arg Arg Ala 325 330 335 Lys Ala Val Ala Lys Met LeuGlu Lys Tyr Gly Val Ser Ala Asp Arg 340 345 350 Ile Thr Ile Glu Trp LysGly Ser Ser Glu Gln Ile Tyr Glu Glu Asn 355 360 365 Ala Trp Asn Arg IleVal Val Met Thr Ala Ala Glu 370 375 380

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidhaving at least 95% identity to the amino acid sequence of SEQ ID NO:2over the entire length of SEQ ID NO:2; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:2, (iii) an isolatedpolypeptide that is the amino acid sequence of SEQ ID NO:2, and (iv) apolypeptide that is encoded by a recombinant polynucleotide comprisingthe polyncleotide sequence of SEQ ID NO:1.
 2. An isolated polynucleotideselected from the group consisting of: (i) an isolated polynucleotidecomprising a polynucleotide sequence encoding a polypeptide that has atleast 95% identity to the amino acid sequence of SEQ ID NO:2, over theentire length of SEQ ID NO:2; (ii) an isolated polynucleotide comprisinga polynucleotide sequence that has at least 95% identity over its entirelength to a polynucleotide sequence encoding the polypeptide of SEQ IDNO:2; (iii) an isolated polynucleotide comprising a nucleotide sequencethat has at least 95% identity to that of SEQ ID NO:1 over the entirelength of SEQ ID NO:1; (iv) an isolated polynucleotide comprising anucleotide sequence encoding the polypeptide of SEQ ID NO:2; (v) anisolated polynucleotide that is the polynucleotide of SEQ ID NO:1; (vi)an isolated polynucleotide of at least 30 nucleotides in lengthobtainable by screening an appropriate library under stringenthybridization conditions with a probe having the sequence of SEQ ID NO:1or a fragment thereof of of at least 30 nucleotides in length; (vii) anisolated polynucleotide encoding a mature polypeptide expressed by theginS gene comprised in the Porphyromonas gingivalis; and (viii) apolynucleotide sequence complementary to said isolated polynucleotide of(i), (ii), (iii), (iv), (v), (vi) or (vii).
 3. A method for thetreatment of an individual: (i) in need of enhanced activity orexpression of or immunological response to the polypeptide of claim 1comprising the step of: administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or (ii) having need to inhibit activity or expression of the polypeptideof claim 1 comprising: (a) administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or (b) administering to the individual a nucleic acid molecule thatinhibits the expression of a polynucleotide sequence encoding saidpolypeptide; (c) administering to the individual a therapeuticallyeffective amount of a polypeptide that competes with said polypeptidefor its ligand, substrate, or receptor; or (d) administering to theindividual an amount of a polypeptide that induces an immunologicalresponse to said polypeptide in said individual.
 4. A process fordiagnosing or prognosing a disease or a susceptibility to a disease inan individual related to expression or activity of the polypeptide ofclaim 1 in an individual comprising the step of: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid polypeptide in an organism in said individual; or (b) analyzing forthe presence or amount of said polypeptide expression in a samplederived from said individual.
 5. A process for producing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO:1, comprising the step of culturinga host cell under conditions sufficient for the production of thepolypeptide.
 6. A process for producing a host cell comprising anexpression system or a membrane thereof expressing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO:1, said process comprising the stepof transforming or transfecting a cell with an expression systemcomprising a polynucleotide capable of producing said polypeptide of(i), (ii), (iii) or (iv) when said expression system is present in acompatible host cell such the host cell, under appropriate cultureconditions, produces said polypeptide of (i), (ii), (iii) or (iv).
 7. Ahost cell or a membrane expressing a polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least 95% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2; (ii) an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO:2; (iii) an isolated polypeptide that is the amino acidsequence of SEQ ID NO:2, and (iv) a polypeptide that is encoded by arecombinant polynucleotide comprising the polynucleotide sequence of SEQID NO:1.
 8. An antibody immunospecific for the polypeptide of claim 1.9. A method for screening to identify compounds that agonize or thatinhibit the function of the polypeptide of claim 1 that comprises amethod selected from the group consisting of: (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution comprising a polypeptide of claim 1,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 10. An agonist or antagonist to thepolypeptide of claim 1.